scholarly journals Mechanistic insight into DsyB/DSYB, key enzymes in marine dimethylsulfoniopropionate synthesis

2021 ◽  
Author(s):  
Jonathan Todd ◽  
Chun-Yang Li ◽  
Jason Crack ◽  
Simone Newton-Payne ◽  
Andrew Murphy ◽  
...  
Keyword(s):  
Marine Algae ◽  
Nucleophilic Attack ◽  
Mechanistic Insight ◽  
Signalling Molecule ◽  
Major Nutrient ◽  
Methyltransferase Gene ◽  
Key Enzyme ◽  
Algae And Bacteria ◽  
Insight Into

Abstract Marine algae and bacteria produce eight billion tonnes of the organosulfur molecule dimethylsulfoniopropionate (DMSP) in Earth’s surface oceans every year. DMSP is an anti-stress compound and, once released into the environment, a major nutrient, signalling molecule and source of climate-active gases. The methionine transamination pathway for DMSP synthesis is used by most known DMSP-producing algae and bacteria. The S-directed S-adenosylmethionine-dependent methyltransferase (SAM-MT) 4-methylthio-2-hydroxybutyrate (MTHB) S-methyltransferase, encoded by the dsyB/DSYB gene, is the key enzyme of this pathway, generating S-adenosylhomocysteine (SAH) and 4-dimethylsulfonio-2-hydroxybutyrate (DMSHB). dsyB/DSYB, present in most DMSP-producing bacteria and haptophyte and dinoflagellate algae with the highest known DMSP concentrations, is shown to be far more abundant and transcribed in marine environments than any other known DMSP synthesis pathway S-methyltransferase gene. Furthermore, we demonstrate in vitro activity of the bacterial DsyB enzyme from Nisaea denitrificans, and provide its crystal structure in complex with SAM and SAH-MTHB, which together provide the first mechanistic insights into a DMSP synthesis enzyme. Structural and mutational analyses imply that DsyB adopts a novel mechanism, distinct from any previously reported SAM-MT, in which the DsyB residue Tyr142 activates the sulfur atom of MTHB for nucleophilic attack on the SAM methyl group. Sequence analysis suggests that this mechanism is common to all bacterial DsyB enzymes and also, importantly, eukaryotic DSYB enzymes from e.g., algae that are the major DMSP producers in Earth’s surface oceans.

scholarly journals SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

2021 ◽  
Author(s):  
Scott B Biering ◽  
Francielle Tramontini Gomes de Sousa ◽  
Laurentia V. Tjang ◽  
Felix Pahmeier ◽  
Richard Ruan ◽  
...  
Keyword(s):  
Transcriptional Response ◽  
Barrier Dysfunction ◽  
Vascular Leak ◽  
Endothelial Barrier Dysfunction ◽  
Mechanistic Insight ◽  
Starting Point ◽  
Signaling Axis ◽  
Insight Into

Severe COVID-19 is associated with epithelial and endothelial barrier dysfunction within the lung as well as in distal organs. While it is appreciated that an exaggerated inflammatory response is associated with barrier dysfunction, the triggers of this pathology are unclear. Here, we report that cell-intrinsic interactions between the Spike (S) glycoprotein of SARS-CoV-2 and epithelial/endothelial cells are sufficient to trigger barrier dysfunction in vitro and vascular leak in vivo, independently of viral replication and the ACE2 receptor. We identify an S-triggered transcriptional response associated with extracellular matrix reorganization and TGF-β signaling. Using genetic knockouts and specific inhibitors, we demonstrate that glycosaminoglycans, integrins, and the TGF-β signaling axis are required for S-mediated barrier dysfunction. Our findings suggest that S interactions with barrier cells are a contributing factor to COVID-19 disease severity and offer mechanistic insight into SARS-CoV-2 triggered vascular leak, providing a starting point for development of therapies targeting COVID-19 pathogenesis.

scholarly journals Histone H3K23-specific acetylation by MORF is coupled to H3K14 acylation

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Brianna J. Klein ◽  
Suk Min Jang ◽  
Catherine Lachance ◽  
Wenyi Mi ◽  
Jie Lyu ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
Memory Impairment ◽  
Target Genes ◽  
Epigenetic Mark ◽  
Mechanistic Insight ◽  
Genomic Analyses ◽  
Insight Into

Abstract Acetylation of histone H3K23 has emerged as an essential posttranslational modification associated with cancer and learning and memory impairment, yet our understanding of this epigenetic mark remains insufficient. Here, we identify the native MORF complex as a histone H3K23-specific acetyltransferase and elucidate its mechanism of action. The acetyltransferase function of the catalytic MORF subunit is positively regulated by the DPF domain of MORF (MORFDPF). The crystal structure of MORFDPF in complex with crotonylated H3K14 peptide provides mechanistic insight into selectivity of this epigenetic reader and its ability to recognize both histone and DNA. ChIP data reveal the role of MORFDPF in MORF-dependent H3K23 acetylation of target genes. Mass spectrometry, biochemical and genomic analyses show co-existence of the H3K23ac and H3K14ac modifications in vitro and co-occupancy of the MORF complex, H3K23ac, and H3K14ac at specific loci in vivo. Our findings suggest a model in which interaction of MORFDPF with acylated H3K14 promotes acetylation of H3K23 by the native MORF complex to activate transcription.

scholarly journals Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy

2019 ◽  
Author(s):  
Tugsan Tezil ◽  
Manish Chamoli ◽  
Che-Ping Ng ◽  
Roman P. Simon ◽  
Victoria J. Butler ◽  
...  
Keyword(s):  
Small Molecules ◽  
Physiological Function ◽  
Nordihydroguaiaretic Acid ◽  
Mechanistic Insight ◽  
Epigenetic Regulator ◽  
Novel Target ◽  
Cellular Thermal Shift Assay ◽  
Insight Into ◽  
In Cells

AbstractAging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a novel target of NDGA and provide mechanistic insight into its role in longevity.

scholarly journals A Trypanosoma brucei orphan kinesin employs a convergent microtubule organization strategy to complete cytokinesis

2021 ◽  
Author(s):  
Thomas E Sladewski ◽  
Paul C Campbell ◽  
Neil Billington ◽  
Alexandra D'Ordine ◽  
Christopher L de Graffenried
Keyword(s):  
Trypanosoma Brucei ◽  
Biophysical Properties ◽  
Microtubule Organization ◽  
Division Plane ◽  
Mechanistic Insight ◽  
Organization Strategy ◽  
Cytoskeletal Elements ◽  
Order Structures ◽  
Insight Into

Many single-celled eukaryotes have complex cell morphologies defined by cytoskeletal elements comprising microtubules arranged into higher-order structures. Trypanosoma brucei (T. brucei) cell polarity is mediated by a parallel array of microtubules that underlie the plasma membrane and define the auger-like shape of the parasite. The subpellicular array must be partitioned and segregated using a microtubule-based mechanism during cell division. We previously identified an orphan kinesin, KLIF, that localizes to the division plane and is essential for the completion of cytokinesis. To gain mechanistic insight into how this novel kinesin functions to complete cleavage furrow ingression, we characterized the biophysical properties of the KLIF motor domain in vitro. We found that KLIF is a non-processive dimeric kinesin that dynamically crosslinks microtubules. Microtubules crosslinked in an antiparallel orientation are translocated relative to one another by KLIF, while microtubules crosslinked parallel to one another remain static, resulting in the formation of organized parallel bundles. In addition, we found that KLIF stabilizes the alignment of microtubule plus ends. These features provide a mechanistic understanding for how KLIF functions to form a new pole of aligned microtubule plus ends that defines the shape of the new posterior, which is a unique requirement for the completion of cytokinesis in T. brucei.

scholarly journals Mitochondrial TRAK adaptors coordinate dynein and kinesin motility

2021 ◽  
Author(s):  
John T. Canty ◽  
Andrew Hensley ◽  
Ahmet Yildiz
Keyword(s):  
Neuronal Activity ◽  
Coiled Coil ◽  
Mitochondrial Transport ◽  
Coordinated Action ◽  
Coiled Coil Domain ◽  
Mechanistic Insight ◽  
Docking Protein ◽  
Insight Into

In neurons, mitochondria are transported to distal regions for supplying energy and buffer calcium. Mitochondrial transport is mediated by Miro and TRAK adaptors that recruit kinesin and dynein-dynactin. To understand how mitochondria are transported by these opposing motors and stalled at regions with elevated calcium levels, we reconstituted the mitochondrial transport machinery in vitro. We show that the coiled-coil domain of TRAK activates dynein-dynactin motility, but kinesin requires an additional factor to efficiently transport Miro/TRAK. Unexpectedly, TRAK adaptors that recruit both motors move towards the plus-end, whereas kinesin is excluded from binding TRAK transported by dynein-dynactin. The assembly and motility of the transport machinery are not affected by calcium. Instead, the mitochondrial docking protein syntaphilin is sufficient to oppose the forces generated by kinesin and stall the motility. Our results provide mechanistic insight into how mitochondria are transported by the coordinated action of motors and statically anchored to regions with high neuronal activity.

scholarly journals Differential expression of MAGEA6 toggles autophagy to promote pancreatic cancer progression

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Yiu Huen Tsang ◽  
Yumeng Wang ◽  
Kathleen Kong ◽  
Caitlin Grzeskowiak ◽  
Oksana Zagorodna ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Progression ◽  
Malignant Tumors ◽  
Nutrient Deficiency ◽  
Ductal Adenocarcinoma ◽  
Mechanistic Insight ◽  
Cancer Initiation ◽  
Cancer Immunotherapeutic ◽  
Insight Into

The melanoma-associated antigen family A (MAGEA) antigens are expressed in a wide variety of malignant tumors but not in adult somatic cells, rendering them attractive targets for cancer immunotherapy. Here we show that a number of cancer-associated MAGEA mutants that undergo proteasome-dependent degradation in vitro could negatively impact their utility as immunotherapeutic targets. Importantly, in pancreatic ductal adenocarcinoma cell models, MAGEA6 suppresses macroautophagy (autophagy). The inhibition of autophagy is released upon MAGEA6 degradation, which can be induced by nutrient deficiency or by acquisition of cancer-associated mutations. Using xenograft mouse models, we demonstrated that inhibition of autophagy is critical for tumor initiation whereas reinstitution of autophagy as a consequence of MAGEA6 degradation contributes to tumor progression. These findings could inform cancer immunotherapeutic strategies for targeting MAGEA antigens and provide mechanistic insight into the divergent roles of MAGEA6 during pancreatic cancer initiation and progression.

scholarly journals De Novo Assessment and Review of Pan-American Pit Viper Anticoagulant and Procoagulant Venom Activities via Kinetomic Analyses

Toxins ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 94 ◽  
Author(s):  
Vance G. Nielsen ◽  
Nathaniel Frank ◽  
Sam Afshar
Keyword(s):  
South America ◽  
Central America ◽  
De Novo ◽  
Inhibit Activity ◽  
North Central ◽  
Mechanistic Insight ◽  
Carrier Molecule ◽  
Pan American ◽  
Insight Into

Snakebite with hemotoxic venom continues to be a major source of morbidity and mortality worldwide. Our laboratory has characterized the coagulopathy that occurs in vitro in human plasma via specialized thrombelastographic methods to determine if venoms are predominantly anticoagulant or procoagulant in nature. Further, the exposure of venoms to carbon monoxide (CO) or O-phenylhydroxylamine (PHA) modulate putative heme groups attached to key enzymes has also provided mechanistic insight into the multiple different activities contained in one venom. The present investigation used these techniques to characterize fourteen different venoms obtained from snakes from North, Central, and South America. Further, we review and present previous thrombelastographic-based analyses of eighteen other species from the Americas. Venoms were found to be anticoagulant and procoagulant (thrombin-like activity, thrombin-generating activity). All prospectively assessed venom activities were determined to be heme-modulated except two, wherein both CO and its carrier molecule were found to inhibit activity, while PHA did not affect activity (Bothriechis schlegelii and Crotalus organus abyssus). When divided by continent, North and Central America contained venoms with mostly anticoagulant activities, several thrombin-like activities, with only two thrombin-generating activity containing venoms. In contrast, most venoms with thrombin-generating activity were located in South America, derived from Bothrops species. In conclusion, the kinetomic profiles of venoms obtained from thirty-two Pan-American Pit Viper species are presented. It is anticipated that this approach will be utilized to identify clinically relevant hemotoxic venom enzymatic activity and assess the efficacy of locally delivered CO or systemically administered antivenoms.

scholarly journals Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Tugsan Tezil ◽  
Manish Chamoli ◽  
Che-Ping Ng ◽  
Roman P. Simon ◽  
Victoria J. Butler ◽  
...  
Keyword(s):  
Small Molecules ◽  
Physiological Function ◽  
Nordihydroguaiaretic Acid ◽  
Mechanistic Insight ◽  
Progressive Loss ◽  
Epigenetic Regulator ◽  
Cellular Thermal Shift Assay ◽  
Insight Into ◽  
In Cells

Abstract Aging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a target of NDGA and provide mechanistic insight into its role in longevity.

Conformational flexibility and allosteric regulation of cathepsin K

2010 ◽  
Vol 429 (2) ◽  
pp. 379-389 ◽  
Author(s):  
Marko Novinec ◽  
Lidija Kovačič ◽  
Brigita Lenarčič ◽  
Antonio Baici
Keyword(s):  
Dermatan Sulfate ◽  
Molecular Level ◽  
Cathepsin K ◽  
Conformational Flexibility ◽  
Kinetic Properties ◽  
Collagenolytic Activity ◽  
Key Enzyme ◽  
Multiple Conformations ◽  
Insight Into

The human cysteine peptidase cathepsin K is a key enzyme in bone homoeostasis and other physiological functions. In the present study we investigate the mechanism of cathepsin K action at physiological plasma pH and its regulation by modifiers that bind outside of the active site. We show that at physiological plasma pH the enzyme fluctuates between multiple conformations that are differently susceptible to macromolecular inhibitors and can be manipulated by varying the ionic strength of the medium. The behaviour of the enzyme in vitro can be described by the presence of two discrete conformations with distinctive kinetic properties and different susceptibility to inhibition by the substrate benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin. We identify and characterize sulfated glycosaminoglycans as natural allosteric modifiers of cathepsin K that exploit the conformational flexibility of the enzyme to regulate its activity and stability against autoproteolysis. All sulfated glycosaminoglycans act as non-essential activators in assays using low-molecular-mass substrates. Chondroitin sulfate and dermatan sulfate bind at one site on the enzyme, whereas heparin binds at an additional site and has a strongly stabilizing effect that is unique among human glycosaminoglycans. All glycosaminoglycans stimulate the elastinolytic activity of cathepsin K at physiological plasma pH, but only heparin also increases the collagenolytic activity of the enzyme under these conditions. Altogether these results provide novel insight into the mechanism of cathepsin K function at the molecular level and its regulation in the extracellular space.

scholarly journals Mechanistic insight into the rapid one-step facile biofabrication of antibacterial silver nanoparticles from bacterial release and their biogenicity and concentration-dependent in vitro cytotoxicity to colon cells

RSC Advances ◽  
2017 ◽  
Vol 7 (64) ◽  
pp. 40034-40045 ◽  
Author(s):  
Suresh K. Verma ◽  
Ealisha Jha ◽  
Babrubahan Sahoo ◽  
Pritam Kumar Panda ◽  
Arun Thirumurugan ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
In Vitro Cytotoxicity ◽  
Bacterial Strains ◽  
Colon Cells ◽  
Mechanistic Insight ◽  
One Step ◽  
Insight Into

Rapid biosynthesis of silver nanoparticles from Gram +ve and Gram −ve bacterial strains and their hiogenicity dependent antibacterial and cytntoxicity.

Sign in / Sign up

Export Citation Format

Share Document